Interactions between various biological macromolecules control protein folding and assembly, DNA packing, protein-DNA interactions, tissue formation and stability, and many other processes. The Unit on Molecular Forces and Structure studies recognition reactions between biological macromolecules with a particular emphasis on pathology of collagen fiber formation in brittle bone disease (Osteogenesis Imperfecta or OI) and other disorders. We recently demonstrated that monomeric collagen slowly melts several degrees below body temperature. Its equilibrium state at body temperature is a random coil rather than triple helix. We found that micro-unfolding of least stable domains triggers self-assembly of fibers where helices are protected from complete unfolding. We are now beginning to look more closely at molecular details of micro-unfolding and at mechanisms relating changes in collagen thermal stability to connective tissue pathology caused by mutations. The most common causes of moderate to lethal OI are glycine substitutions and deletions or insertions of small fragments in the (Gly-X-Y)n sequence of collagen triple helix. Many researchers reported changes in the thermal stability of collagen accompanying these mutations. However, thermal stability of mutant collagens was previously studied exclusively by the chymotrypsin/trypsin digestion assay. By comparing a variety of techniques and careful analysis of denaturation kinetics we demonstrated that while this assay yields useful information on rapid collagen micro-unfolding it cannot be used for reliable evaluation of changes in overall thermal stability of the protein. From systematic analysis of thermal stability of mutant collagens from NIH collection of OI patients by differential scanning calorimetry and isothermal circular dichroism we found that the same Gly substitution may produce substantially different effect on the melting temperature depending on its location. The position of the mutation within certain domains appears to be more important than the identity of the substituting residue or its immediate local environment. Mutations at the N-terminal end of the molecule appear to cause particularly strong changes in the thermal stability. Furthermore, they have different effect on the melting temperature of procollagen and collagen, which is not the case for mutations located in the middle of the triple helix. We revisited the role of a2(I) in the thermal stability of collagen in osteogenesis imperfecta murine (oim) using purified tendon collagen from wild type (a1(I)2a2(I) - heterotrimers) and oim (a1(I)3) mice as well as artificial a1(I)3 homotrimers obtained by refolding of rat tail tendon collagen. In contrast to earlier reports, we established that oim homotrimers melt at ~ 2 oC higher temperature than wild type heterotrimers, when measured at the same heating rate by differential scanning microcalorimetry. We also found that former melt ~ 100 times slower than latter, when measured at the same temperature by isothermal circular dichroism. Detailed analysis of microunfolding kinetics at different temperatures by proteolytic cleavage and gel electrophoresis revealed that: (a) Initial microunfolding of oim homotrimers and wild type heterotrimers occurs at approximately the same rate but at different sites. (b) The weakest spot on oim triple helix appears to be about 100 amino acids from the C-terminal end within the cyanogen bromide peptide CB6, consistent with the previous finding. (c) However, we found that the same microunfolding site is also present in wild type collagen, although the weakest spot of the latter is located close to the N-terminal end of CB8. (d) Overall, the patterns of microunfolding sites in type I hetero- and homo-trimers are substantially different. Amino acid analysis and differential gel electrophoresis showed that the observed changes are associated with difference in the amino acid composition of a1(I) and a2(I) chains rather than with posttranslational modification. We extended structural and functional studies of collagen from mice with G349C substitution. We observed that mutant molecules are equally well incorporated into the matrix as normal collagen helices and they form mature covalent crosslinks with the same efficiency.We detected only minimal posttranslational overmodification of mutant molecules in cell cultures and in animal tissues. The extent of posttranslational modification was similar in lethal and non-lethal animals but varied between different tissues. We found no substantial changes in the thermal stability, rate of thermal denaturation, in vitro fibrillogenesis or intermolecular forces between mutant collagens in fibers. Unlike many other reported Gly-Cys mutations, this substitution appears to be remarkably well compensated. The only substantial molecular pathology we were able to detect was a disruption of quasi-crystalline lateral packing of helices in tendons that appears to be associated with abnormal collagen interaction with other matrix components, most likely glycosaminoglycans. Abnormal collagen interactions with glycosaminoglycans and proteoglycans may cause fibril disorganization in bone observed in some of the animals and result in bone fragility. Verification of this hypothesis is the focus of our present studies. We investigated biochemical consequences of realignment of X and Y position interactions along the helix resulting from Gly-Ala-Hyp triplet duplication at AA 868-876. We previously reported a decrease in the melting temperature of ~ 2 oC for helices with one mutant chain and ~ 6 oC for helices with two mutant chains. In the last year we found that N-propeptide cleavage is slower in collagen with the triplet duplication than in controls or procollagen with an a1(I) G832S substitution, showing that the register shift persists through the entire helix. The register shift causes substantially faster cleavage of mutant protein by mammalian collagenase and abnormal cleavage by chymotrypsin and trypsin at sub-melting temperatures indicating altered local micro-unfolding pattern. Apparently the mutation causes a substantial reduction in the thermal stability of the C-terminal end of the molecule. The register shift also disrupts incorporation of mutant collagen into fibrils and matrix. Only 30% of mutant monomers incorporated into fibrils. The fibrils which did form contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. Since double mutant molecules were most severely impaired, alignment of the a2(I) chain appears to be critical. Both collagen and Mn2+-DNA self-assemble into fibers at elevated temperature. Traditionally, such counterintuitive temperature dependence is attributed to the entropy gain associated with the release of structured water upon fiber assembly. However, our work showed that in collagen the temperature dependence is related to the entropy gain upon triple helix micro-unfolding in fibers. We, thus, reevaluated potential mechanisms of the temperature dependence in DNA aggregation. We developed a theoretical model which showed that the observed temperature dependence in DNA condensation may be associated with Mn2+ repartitioning between binding sites in minor and major grooves.